Adeno-associated virus production platform

Abstract

Provided herein is a novel rAAV-based HSV production method comprising two engineered cell lines: an engineered CHO cell for the production of a plurality of AAV serotypes and an engineered BHK-21 for the production of an rHSV-1 stock solution, which is used for the production of rAAV in CHO cells. The developed method provides a scalable serum-free manufacturing platform.

Description

Background Art

[0001] Recombinant adeno-associated viral vectors (rAAV) are the leading platform for gene delivery, with three licensed products currently approved as of December 2021 (Bulcha et al., 2021). rAAV has several advantages as a gene delivery vector, including their ability to transduce a variety of proliferating and non-proliferating cells, accommodate cell / tissue-specific promoters, and elicit a blunted immune response compared to other viral vectors (Kang et al., 2009). AAV is a small, non-enveloped virus in the Parvoviridae family of the genus Dependovirus (Srivastava et al., 1983; Daya and Berns, 2008). The AAV 4.6-kb single-stranded DNA genome contains two viral genes: rep and cap. These genes can be removed and replaced with a cassette expressing a therapeutic transgene and the essential rep and cap genes provided in trans (Becerra et al., 1988). The AAV capsid is icosahedral and assembled from 60 viral protein (VP) monomers, with approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3 (Van Vliet et al., Methods Mol. Biol. 2008; 437: 51-91.)

[0002] Currently, rAAV has different cell culture expression platforms, including stable packaging cell lines expressing the rep and cap genes of the desired rAAV serotype, stable proviral cell lines stably expressing Rep, Cap and transgenes, and triple transient transfection (Clark et al., 1995, Clark., 2002, Qiao et al., 2002). Triple transient transfection is the most common method for producing rAAV, which uses three plasmids: one encoding the gene of interest (GOI) flanked by AAV inverted terminal repeats (ITRs), the second encoding the AAV rep and cap genes, and the third encoding adenoviral helper function genes. By including helper genes in the rep-cap plasmid, this three-plasmid system can be simplified to co-transfection (Grimm et al., 1998, and Clark K.R., 2002). However, the triple transient transfection method is challenging to scale up to very high doses, which result in low specific yields of infectious particles (ip) and often high DNase-resistant particle (DRP / ip) ratios (50-100) (Grimm et al., Hum Gene Ther; 9: 2745-2760 (1998) and Zolotukhin et al., 2002 Methods; 28: 158-167).

[0003] As with adenovirus, recombinant HSV-1 (rHSV-1) has been reported to support rAAV replication as part of a helper virus system (Buvler et al., 1981 J Virol; 40: 241-247). The minimal HSV helper genes required for rAAV replication are the HSV helicase-primase complex (UL5, UL8, UL52), HSV DNA polymerase, and HSV DNA binding protein (UL29) genes (Weindler and Heilbronn., 1991 J Virol; 65: 2476-2483). In addition, a rHSV co-infection approach using two HSV-1 vectors (one encoding the desired rAAV rep / cap serotype and the second encoding the GOI) has been reported to produce very high specific yields of multiple different serotypes of rAAV in various cell lines with very low (DRP / ip) ratios (Kang et al., 2009). Another advantage of the HSV helper system is that HSV can replicate and provide helper function in different mammalian cells, which means that HSV helper function is not restricted by the host range of rAAV production, in contrast to the requirement of human cell lines for successful adenovirus-assisted rAAV production (Buller et al., 1970 J Gen Virol; 43: 663-672).

[0004] Chinese hamster ovary (CHO) cells are primarily used as expression hosts for recombinant monoclonal antibody (mAb) production and other therapeutic protein production, which constitute the fastest growing segment of the biopharmaceutical industry (Walsh G., 2018 Nat. Biotechnol; 36: 1136-1145). The use of CHO cells for viral production is desirable due to their regulatory acceptability and low manufacturing costs relative to human cells. However, there is limited experience (if any) in the literature regarding the use of CHO cells for rAAV production, likely due to cellular restriction factors in these cells that may affect rAAV production and interfere with viral packaging. For example, CHO cells have been shown not to support vaccinia virus replication during viral intermediate protein synthesis (Ramsey-Ewing and Moss. 1995 Virology. 1995 Feb 1; 206(2): 984-93). In addition, CHO cells are naturally resistant to HSV-1 productive infection due to the lack of key receptors for HSV-1 entry and infection (Montgomery et al., 1996). Therefore, there is a need in the art to engineer serum-free, adaptive suspension CHO CAT-S cells to allow infection with replication-defective HSV-1 vectors encoding the essential elements for the production of different rAAVs. Summary of the Invention

[0005] The present disclosure relates to baby hamster kidney (BHK) cells adapted for growth under serum-free conditions, wherein the cells stably express a hamster codon-optimized herpes simplex virus-1 (HSV-1) ICP27 open reading frame comprising a deletion of a non-essential infectious cell protein 27 (ICP27) element. In one aspect, the cells are grown in suspension. In another aspect, the non-essential ICP27 elements are the 5' and 3' untranslated regions (UTRs).

[0006] The present disclosure also relates to cell lines comprising the BHK cells described herein.

[0007] The present disclosure also relates to a method for producing a recombinant adeno-associated virus (rAAV) vector, which comprises introducing a recombinant herpes virus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a target gene into the BHK cells or cell lines described herein; and culturing the cells or cell lines under conditions for producing rAAV vectors.

[0008] The present disclosure also relates to Chinese hamster ovary (CHO) cells suitable for growth under serum-free conditions, wherein the cells stably express one or more polypeptides necessary for invasion and infection by herpes simplex virus-1 (HSV-1). In one aspect, the CHO cells stably express herpes virus entry mediator (HVEM) and / or nectin-1. In another aspect, the HVEM and / or nectin-1 sequences have been codon-optimized for expression in CHO cells.

[0009] The present disclosure also relates to the CHO cells described herein.

[0010] The present disclosure also relates to a method for producing a recombinant adeno-associated virus (rAAV) vector, the method comprising introducing a recombinant herpes virus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a gene of interest (GOI) into a CHO cell or cell line described herein; and culturing the cell or cell line under conditions for producing rAAV vectors. In another aspect, the rHSV vector is introduced at a multiplicity of infection of rHSV-rep / cap:rHSV-GOI of about 4:1, 6:1, 8:1, or 10:1. In another aspect, the AAV serotype is AAV6, AAV8, or AAV9. In another aspect, the gene of interest encodes any therapeutic biological compound. In another aspect, the therapeutic biological compound is an antibody or a chimeric antigen receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the plasmid construction used for stable transfection and the generation of eight pools. Figure 1a shows four separate cassettes; Pool 1 (CMV-HVEM), in which the HVEM ORF is constructed under the CMV promoter and upstream SV40 polyadenylation. Pool 2 (CMV-HVEM-CO), in which the codon-optimized HVEM ORF is constructed under the CMV promoter and upstream SV40 polyadenylation. Pool 3 (CMV-Nectin-1), in which the Nectin-1 ORF is constructed under the CMV promoter and BGH polyadenylation. Pool 4 (CMV-Nectin-1-CO), in which the codon-optimized Nectin-1 ORF is constructed under the CMV promoter and BGH polyadenylation. Figure 1b shows four double cassettes; Pool 5 (CMV-HVEM-Nectin-1), in which the HVEM and Nectin-1 ORFs were constructed and flanked by the CMV promoter and SV40 polyadenylation and the CMV promoter and BGH polyadenylation, respectively. Pool 6 (CMV-HVEM-Nectin-1-CO), in which codon-optimized HVEM and Nectin-1 ORFs were constructed under the CMV promoter and upstream SV40 polyadenylation and the CMV promoter and upstream BGH polyadenylation, respectively. Pool 7 (Spro-HVEM-Nectin-1), in which the HVEM and Nectin-1 ORFs were constructed under a synthetic promoter (Spro) and upstream SV40 polyadenylation and Spro and BGH polyadenylation, respectively. Pool 8 (Spro-HVEM-Nectin-1-CO) was constructed in which codon-optimized HVEM and Nectin-1 ORFs were constructed and flanked by Spro and SV40 polyadenylation and Spro and BGH polyadenylation, respectively. Figure 1c shows that high levels of HVEM surface expression from pools (CMV-HVEM, CMV-HVEM-CO, CMV-HVEM-Nectin-1, CMV-HVEM-Nectin-1-CO, Spro-HVEM-Nectin-1, and Spro HVEM-Nectin-1-Co) were detected and expressed as mean fluorescence intensity (MFI). Figure 1d High levels of Nectin-1 surface expression from the pools (CMV-Nectin-1, CMV-Nectin-1-CO, CMV-HVEM-Nectin-1, CMV-HVEM-Nectin-1-CO, Spro-HVEM-Nectin-1, and Spro HVEM-Nectin-1-Co) were detected and are shown as MFI.

[0012] Figure 2 shows green fluorescent protein (GFP) expression and rAAV9-GFP production from stable CHO cell pools. Figure 2a shows the average GFP expression of stable cell pools (MOI=10) infected with rHSV-nols-AAV-GFP at a total of six time points (12, 24, 36, 48, 72 and 96) after infection. Pools 1 and 2 outperformed all other pools and infected hosts. All pools outperformed wild-type host-infected CHO (p<0.0001). Figure 2b shows the qPCR rAAV9-GFP titer (vg / mL) of the supernatant from the infected stable cell pool 24hpi using rHSV-nols-AAV-GFP and rHSV-AAV9 vectors at MOI 1:1. No significant differences were observed in the rAAV9-GFP physical titers produced by the eight test pools. All co-infected cell pools produced higher rAAV9-GFP physical titers compared to cells infected with wild-type hosts. No significant differences in rAAV9-GFP titers were detected among the pools tested (p=0.0976).All samples were tested in duplicate and all data are expressed as mean±SD.

[0013] Figure 3 Shown are the generation of rHSV-GFP infected high and medium CHO-HVEM expressing clones analyzed by flow cytometry.

[0014] Fig. 4 shows the characterization and test of selected CHO-HVEM expression clone for rHSV-1-GFP infection.Fig. 4 a shows the recovery clones of final 24 selections, and they have high HVEM expression (expressed as MFI).Ten of these clones are high HVEM expression clones (clones 1,7,21,23,24,33,36,40,63 and 64).Other remaining 14 clones are middle HVEM expression clones (clones 9,11,13,14,15,16,23,28,29,42,46,51,54,62).Fig. 4 b shows the rHSV-1GFP vector invasion and infection using IncuCyte to all 24 clones and retest (record two time points).All infected clones show high GFP expression after infection.However, no significant difference (p=0.5251) was observed in the clones tested for GFP expression.

[0015] Figure 5 shows the production of rAAV6.2-GFP in eight selected CHO-HVEM clones. Figure 5a shows the decrease in cell viability after rHSV-1 vector co-infection. Figure 5b shows the decrease in viable cell density after rHSV-1 vector co-infection. Figure 5c shows the rAAV6.2-GFP titer from eight tested CHO-HVEM expression clones using an MOI of 1:1. Figure 5d shows the production of rAAV6.2-GFP in CHO-HV-C1 and CHO-HV-C62 clones using different MOIs. Figure 5e shows the production of rAAV8-GFP and rAAV9-GFP in CHO-HV-C1 using an MOI of 4:1.

[0016] Figure 6 Shown are the cell viability after CHO-HV-C1 co-infection ( Figure 6 a) and rAAV6.2GFP vector production ( Figure 6 b) is a significant improvement.

[0017] Figure 7 Schematic diagram showing the process of harvesting and purifying rAAV vectors using the PEG-chloroform method.

[0018] Figure 8 shows the analytical characterization of rAAV produced in the CHO-HV-C1 clone. Figure 8a shows good expression of VP1, VP2 and VP3 capsid proteins, which are derived from purified rAAV6.2-GFP (produced using different MOIs; 4:2, 6:2, 8:2 and 10:2) and rAAV9-GFP vectors (produced using different MOIs; 4:2, 6:2 and 8:2). Figure 8b shows a miniature transmission electron micrograph (miniTEM) of purified rAAV6.2-GFP, which shows 91% complete capsids, no aggregates and no cell debris. Figure 8c shows a miniTEM of purified rAAV9-GFP, which shows 79.5% complete capsids. White arrows are used to indicate complete capsids, while black arrows are used to indicate incomplete (empty) capsids. FIG8 d shows rAAV capsid ratio detection using the CE-SDS method and shows the absorbance values of VP1, VP2, and VP3 from test rAAV6.2-GFP and rAAV9-GFP produced in the CHO-HV-C1 clone, where impurity detection was performed.

[0019] Figure 9Figure 2 shows the residual infectious rHSV-1 in purified rAAV tested on V27 cells. Complemented V27 cells stably expressing the HSV-1 ICP27 protein were tested for any infectious residual of the rHSV-1 vector used to produce rAAV in the CHO-HV-C1 clone. No cytopathic effect (CPE) was observed in the wells inoculated with PEG-chloroform purified rAAV6.2-GFP and / or rAAV9-GFP vectors 2 to 4 days after infection, indicating that the rHSV-1 vector used for co-infection was completely inactivated compared to the wells inoculated with rHSV-AAV-GFP vectors, where clear CPE appeared to indicate cell rounding and detachment of cell membrane sheets.

[0020] Figure 10 shows the infectivity of rAAV produced in CHO-HV-C1 cells. Figure 10a shows the infectivity of purified rAAV6.2-GFP vectors produced in the CHO-HV-C1 clone and purified using the PEG-chloroform method and tested on Ad293 cells, and is compared with the infectivity of rAAV6.2-GFP vectors produced in HEK293 cells and purified by chromatography. Figure 10b shows the infectivity of PEG-chloroform-purified rAAV9-GFP vectors produced in the CHO-HV-C1 clone and tested on Ad293 cells, and is compared with the infectivity of rAAV9-ZsGreen vectors produced in HEK293 cells and purified by chromatography. Figure 10c shows in vitro transduction of rAAV produced in CHO-HV-C1 cells. Transduction of rAAV6.2 and rAAV9-GFP produced in CHO cells and purified by PEG-chloroform was compared with transduction of rAAV6.2-GFP and rAAV9-ZsGreen produced in HEK293 cells and purified by chromatography.

[0021] Figure 11 shows the biodistribution of rAAV derived from CHO cells. Figure 11a shows the experimental design. Mice were divided into five groups. G1, G2, G3, and G4 were treated with 10 11vg / 100 μL of rAAV6.2-GFP-CHO, rAAV9-GFP-CHO, rAAV6.2-GFP from triple transient transfection (TTT), or rAAV9-ZsGreen from TTT. G5 was inoculated with sterile PBS. Three weeks after tail vein injection, all mice were euthanized, and tissues (liver, heart, lung, kidney and skeletal muscle) were harvested and tested for GFP copies by qPCR and expressed by confocal microscopy. Figure 11b shows the GFP titer from G1 and G3 tissues using qPCR. Figure 11c shows the GFP / ZsGreen titer from G2 and G4 tissues using qPCR. Figure 11d shows the GFP expression of liver sections from all groups using confocal microscopy.

[0022] Figure 12 shows the development and selection of HSV-1 producer CHO-ICP27 pool and clone. Figure 12a shows the development of stable CHO-HV1-ICP27 pool using random integration. The HSV-1ICP27ORF optimized by Chinese hamster codon is subcloned into a homemade plasmid for cell line development under CMV promoter and upstream SV40 polyadenylic acid. The puromycin box encoded by the same plasmid is composed of CMV promoter downstream of puromycin ORF and BGH polyadenylic acid. Figure 12b shows the development of stable CHO-HV1-ICP27 pool using CRISPR / Cas9 technology. Donor plasmid is constructed using the puromycin box of the Chinese hamster ICP27ORF downstream CMV promoter and upstream SV40 polyadenylic acid and flank SV40 promoter and SV40 polyadenylic acid of homemade pCLD plasmid backbone and encoding. Two cassettes are flanked by right homology arm and left homology arm (each 750 base pairs). The total length of the two cassettes was 4.1 kb. Figure 12c shows the mean fluorescence intensity (MFI) of ICP27 expression from the final selected clones. Seven clones were site-integrated, such as C6-S, and 17 clones were randomly integrated, such as C-11R.

[0023] Figure 13 The production of rHSV-AAV9 in CHO-HV1-ICP27-C11 is shown. Three different MOIs were used to test the production of rHSV-AAV9 in selected CHO-HV1-ICP27-C11 clones using homemade culture medium at 33°C. The harvested rHSV-AAV9 was titrated by plaque assay on V27 cells.

[0024] Figure 14 Shown is the construction of the BHK-21-ICP27 expression pool. Figure 14 a shows pool 1 expressing the codon-optimized ICP27 ORF under the HSV-1 ICP27 endogenous promoter with puromycin as a selection marker. Figure 14 b shows pool 2 expressing the codon-optimized ICP27 ORF under the CMV promoter with puromycin as a selection marker. Figure 14 c shows pool 3 expressing the non-codon-optimized ICP27 ORF under the CMV promoter with neomycin as a selection marker.

[0025] Figure 15 The production of HSV-AAV6.2 in BHK-21-ICP27 pools is shown. Recovered BHK21-ICP27 pools were tested for rHSV-AAV6.2 production (MOI 0.15 PFU / mL) using Xell HEK TF in the absence of serum or in the presence of 4% FBS. Purified virus was titrated on V27 cells by plaque assay on day 3 post-infection.

[0026] Figure 16 Silver staining results of impurities from PEG-chloroform purified rAAV samples are shown. DETAILED DESCRIPTION

[0027] Recent studies have shown that the use of recombinant herpes simplex virus 1 (rHSV-1) vectors to produce different recombinant adeno-associated viruses (rAAVs) yields rAAVs with higher physical and transduction titers than rAAVs produced by commonly used triple transient transfection methods and / or baculovirus-based systems. However, rAAV-based HSV production platforms currently face two major challenges: (1) reliance on serum-supplemented commercial culture media for high productivity, resulting in prohibitively expensive large-scale production and a high risk of introducing exogenous factors into the final product, and (2) the challenge of scaling up the production of rHSV-1 vectors in adherent Vero cells (e.g., the V27 cell line) that express the HSV-1 ICP27 protein. To address the first challenge, the present disclosure provides eight serum-free, adapted CHO cell pools that express receptors (HVEM and / or Nectin-1) necessary for HSV-1 entry and infection. Using a high-throughput approach, the present disclosure provides a top HVEM receptor-expressing clone (referred to as CHO-HV-C1). Interestingly, 10 log was reported within 48-72 hours after co-infection with a higher MOI. 10 Compared to non-CHO-based platforms, higher yields of rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors were achieved with lower multiplicities of infection (MOIs) within 24 h after co-infection in CHO-HV-C1 clones, with titers of ∼9.21 log, ∼1.07 log, and ∼2.11 log, respectively. 10 , 9.40log 10 and 9.61log 10Viral genomes / mL (vg / mL) (Kang et al., 2009. Gene Therapy 16, 229-239). In addition, rAAV produced in the CHO-HV-C1 clone has in vitro and in vivo transduction efficacy comparable to those produced by triple transient transfection. To address the second challenge, the present disclosure provides a homemade serum-free suspension BHK-21 cell pool engineered to express HSV-1 ICP27 protein. Interestingly, the BHK-21-ICP27 expression pool grown in serum-free medium produces rHSV-1 titers comparable to those of V27 cells.

[0028] Gene therapy has a very promising potential for treating a variety of different diseases that currently have no viable treatment options, such as cystic fibrosis, heart failure and Duchenne muscular dystrophy. In recent years, gene therapy has focused on the use of rAAV vectors because they are expressed for a long time after delivery to the target organ and they are non-pathogenic in humans. The current manufacturing methods of rAAV for clinical research include triple transient transfection (the most common method), packaging or production cell lines and helper virus systems, such as those using HSV-1, baculovirus and / or human adenovirus-5. Transfection-based methods such as triple transient transfection are difficult to expand production and result in low specific yields of infectious particles (ip) and DNase-resistant particles (Grimm et al., 1998. Hum Gene Ther; 9: 2745-2760; Zolotukhin et al., 2002. Methods; 28: 158-167). On the other hand, packaging or production cell lines are limited to producing one rAAV serotype / product. Recent studies have shown that rHSV-1-assisted rAAV production provides an efficient manufacturing method (Kang et al., 2009). However, typical production of rAAV using the rHSV-1 system involves infecting virus producer cells such as BHK-21 cells with two replication-defective rHSV-1 vectors in serum-supplemented medium, which is very expensive for large-scale production and may introduce exogenous viral factors and / or prions into the final product. Another disadvantage of using the rHSV-1 system to manufacture rAAV is the challenge of scaling up the production of rHSV-1 vector stocks, as current production of rHSV-1 vector stocks depends on the use of adherent Vero cells expressing the HSV-1 ICP27 protein (called V27 cells, Rice and Knipe J Virol. 1990 Apr;64(4):1704-15).

[0029] CHO cells are the main mammalian cell type for the production of recombinant protein biologics because they have the ability to correctly fold, assemble, and modify recombinant proteins (Aggarwal, 2014. Nat. Biotechnol. 32, 32-39; Jayapal et al., 2007. Cell Engineering Progress, 103, 40-47; and Walsh, 2018. Nat. Biotechnol; 36: 1136-1145). Certain animal cell types, such as porcine testis (ST) and CHO cells, can effectively bind HSV-1 virus but limit viral entry (Shieh et al., 1992. J. Cell Biol. 116, 1273-1281; Subramanian et al., 1994. J. Virol. 68, 5667-5676). In addition, it has been reported that pig and CHO cells become susceptible to HSV-1 invasion when expressing human cDNA encoding HVEM (Montgomery et al., 1996. Cell Vol. 87, 427-436). The present disclosure aims to use the HSV-1 helper system (a virus that does not naturally infect wild-type CHO cells) to engineer suspended, serum-free, adapted CHO cells to produce different rAAVs.

[0030] rAAV is the dominant platform for gene delivery, with three licensed products approved by the end of 2021 (Bulcha et al., 2021). rAAV has several advantages as a gene delivery vector, including their ability to transduce a variety of proliferating and non-proliferating cells, accommodate cell / tissue-specific promoters, and elicit a blunted immune response compared to other viral vectors (Kang et al., 2009).

[0031] Currently, rAAV has different cell culture expression platforms, including stable packaging cell lines expressing the rep and cap genes of the desired rAAV serotype, stable proviral cell lines stably expressing Rep, Cap, and the transgene, and triple transient transfection (Clark et al., 1995. Hum Gene Ther; 6: 1329-1341, Clark., 2002. Kidney Int; 61s: 9-15, Qiao et al., 2002. Kidney Int; 61s: 9-15). Triple transient transfection is the most common method for producing rAAV, which uses three plasmids: one encoding the gene of interest (GOI) flanked by AAV inverted terminal repeats (ITRs), a second encoding the AAV rep and cap genes, and a third encoding adenoviral helper function genes. This three-plasmid system can be simplified to co-transfection by including helper genes in the rep-cap plasmid (Grimm et al., 1998, and Clark KR, 2002). However, the triple transient transfection method is challenging to scale up to very high doses, which result in low specific yields of infectious particles (ip) and often high DNase-resistant particle (DRP / ip) ratios (50-100) (Grimm et al., 1998. Hum Gene Ther; 9: 2745-2760; Zolotukhin et al., 2002. Methods; 28: 158-167).

[0032] As with adenovirus, recombinant HSV-1 (rHSV-1) has been reported to support rAAV replication as part of a helper virus system (Buller et al., 1981). The minimal HSV helper genes required for rAAV replication are the HSV helicase-primase complex (UL5, UL8, UL52), HSV DNA polymerase, and HSV DNA binding protein (UL29) genes (Weindler and Heilbronn., 1991. J Virol; 65: 2476-2483). In addition, a rHSV co-infection approach using two HSV-1 vectors (one encoding the AAV2 rep and cap of the desired AAV and the second encoding the GOI) has been reported to produce very high specific yields of multiple different serotypes of rAAV in various cell lines with very low (DRP / ip) ratios (Kang et al., 2009). Another advantage of the HSV helper system is that HSV can replicate and provide helper function in different mammalian cells, which means that HSV helper function is not restricted by the host range of rAAV production, compared to adenovirus-assisted rAAV production which requires human cell lines (Buller et al., 1970. J Gen Virol; 43: 663-672).

[0033] Chinese hamster ovary (CHO) cells are primarily used as expression hosts for the production of recombinant monoclonal antibodies (mAbs) and therapeutic proteins, which constitute the fastest growing segment of the biopharmaceutical industry (Walsh G., 2018). Due to the regulatory acceptability and low manufacturing costs of CHO cells relative to human cells, the use of CHO cells for viral production is desirable. However, there is limited experience (if any) in the literature regarding the use of CHO cells for rAAV production, possibly due to the fact that cell restriction factors in these cells may affect rAAV production and interfere with viral packaging. For example, it has been shown that CHO cells do not support vaccinia virus replication during the viral intermediate protein synthesis stage (Ramsey-Ewing and Moss. Virology. 1995 Feb 1; 206 (2): 984-93). In addition, due to the lack of key receptors for non-replicating HSV-1 infection, CHO cells are naturally resistant to HSV-1 productive infection (Montgomery et al., 1996. Cell Vol. 87, 427-436). The present disclosure provides serum-free, adaptive suspension CHO-CAT-S cells engineered to allow HSV-1 entry and infection for the production of various rAAVs using the HSV-1 system.

[0034] definition

[0035] In order to make it easier to understand the present disclosure, some terms are first defined. As used in this specification, unless otherwise explicitly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0036] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "feed medium" should be understood to mean one or more feed media. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0037] As used herein, the term "and / or" should be understood as specifically disclosing each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] It should be understood that wherever aspects are described herein with the language "comprising," other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary for those of skill in the art of many of the terms used in this disclosure.

[0040] Units, prefixes and symbols are expressed in their International System of Units (SI) recognized form. Numerical ranges include numerical values that limit the range. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0041] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0042] The term "about" or "substantially comprising" means that a value or composition is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" can mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms can mean values of up to an order of magnitude or up to 5 times. When a particular value or composition is provided in the application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value or composition.

[0043] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range, and where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0044] The terms "adeno-associated virus," "AAV virus," "AAV virion," "AAV viral particle," and "AAV particle" are used synonymously herein to refer to viral particles composed of at least one capsid protein of AAV and an encapsidated polynucleotide corresponding to the AAV genome. Wild-type AAV refers to a virus belonging to the family Parvoviridae of the genus Dependovirus. The wild-type AAV genome is approximately 4.7 kb in length and consists of single-stranded deoxyribonucleic acid (ssDNA), which can be positive or negative sense. The wild-type genome includes inverted terminal repeats (ITRs) and three open reading frames (ORFs) at both ends of the DNA strand. ORF rep encodes the four Rep proteins essential for the AAV life cycle. ORF cap contains nucleotide sequences encoding the capsid proteins VP1, VP2, and VP3, which interact to form an icosahedral symmetrical capsid. Finally, the assembly activation protein (aap) ORF, which overlaps with the cap ORF, encodes the AAP protein that appears to promote capsid assembly. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell) flanked by AAV ITRs, it is typically referred to as an "AAV vector particle" or "AAV viral vector" or "AAV vector" or "recombinant AAV vector." The invention also encompasses the use of double-stranded AAV or self-complementary AAV (also known as dsAAV or scAAV).

[0045] "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is usually referred to as a "rAAV vector particle" or simply "rAAV vector."

[0046] "Packaging" refers to the series of intracellular events that result in the assembly of capsid proteins and encapsidation of the vector genome to form AAV particles.

[0047] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus, respectively. They have been found in all tested AAV serotypes and are described below and in the art. AAV rep and cap are referred to herein as AAV "packaging genes."

[0048] As used herein, the term "hybrid AAV" refers to an AAV that comprises the capsid protein of one AAV serotype and genomic material from another AAV serotype.

[0049] As used herein, the term "chimeric AAV" refers to an AAV that comprises gene sequences and / or protein sequences derived from two or more AAV serotypes, and may include mutations made to the gene sequences of those two or more AAV serotypes. Exemplary chimeric AAVs may comprise a chimeric AAV capsid, e.g., a capsid protein having one or more amino acid regions derived from two or more AAV serotypes.

[0050] As used herein, the term "AAV variant" refers to an AAV that comprises one or more amino acid mutations in its genome or protein compared to its parent AAV, for example, one or more amino acid mutations in its capsid protein compared to its parent AAV.

[0051] The term "viral vector" refers to a gene transfer vector or gene delivery system derived from a virus. Such vectors can be constructed using recombinant techniques known in the art. In some aspects, the virus used to derive such vectors is selected from AAV, helper-dependent adenovirus, hybrid adenovirus, Epstein-Barr virus, retrovirus, lentivirus, herpes simplex virus, Sendai virus (hemaglutinating virus of Japan, HVJ), Moloney murine leukemia virus, poxvirus, and HIV-based viruses.

[0052] As used herein, the term "AAV virion" or "AAV particle" refers to a viral particle comprising a capsid comprising at least one AAV capsid protein that encapsidates an AAV vector as described herein, wherein in some embodiments, the vector may further comprise a heterologous polynucleotide sequence or a transgene.

[0053] As used herein, the term "engineered cell" and its grammatical equivalents refer to a cell that contains at least one nucleic acid alteration within the cell's genome or contains at least one exogenous nucleic acid or protein. Alterations include additions, deletions, and / or substitutions within a nucleic acid sequence. Thus, engineered cells include cells that contain added, deleted, and / or altered genes.

[0054] Various aspects of the disclosure are described in more detail in the following subsections.

[0055] Adeno-Associated Virus (AAV)

[0056] Adeno-associated virus (AAV) is a non-pathogenic, single-stranded DNA parvovirus. AAV has a capsid diameter of approximately 20 nm. Each end of the single-stranded DNA genome contains inverted terminal repeats (ITRs), which are the only cis-acting elements required for genome replication and packaging. The AAV genome carries two viral genes: rep and cap. The virus utilizes two promoters and alternative splicing to produce the four proteins required for replication (Rep78, Rep 68, Rep 52, and Rep 40). The third promoter produces transcripts of three structural viral capsid proteins 1, 2, and 3 (VP1, VP2, and VP3) through a combination of alternative splicing and alternate translation start codons (Berns KI et al., Bioessays. 1995; 17: 237-45). The three capsid proteins share the same C-terminal 533 amino acids, while VP2 and VP1 contain other N-terminal sequences of 65 and 202 amino acids, respectively. AAV virions contain a total of 60 copies of VP1, VP2, and VP3, which have been observed in crude extracts at a ratio of 1:1:5 (Aucoin MG et al., Biotechnol Adv. 2008; 26(1): 73-88) or in the range of 1:1:8 to 1:1:20 by densitometry (Grimm D et al., Gene Ther, 1999; 6(7): 1322-1330; Kronenbera S et al., EMBO Rep. 2001; 2(11): 997-1002), arranged with T=1 icosahedral symmetry (Rose JA et al., J Virol. 1971; 8: 766-70). AAV requires adenovirus (Ad), herpes simplex virus (HSV) or other viruses as helper viruses to complete its lytic life cycle (Atchison RW et al., Science. 1965; 149: 754-6; Hoggan MD et al., Proc Natl Acad Sci US A. 1966; 55: 1467-74). In addition, in the absence of a helper virus, wild-type (wt) AAV integrates into the chromosome through the interaction of ITRs with the help of Rep proteins to achieve a latent state (Berns et al., 1995).

[0057] AAV serotypes

[0058] There are many different AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and rh-AAV-10. In vivo studies have shown that various AAV serotypes exhibit different tissue tropism or cell tropism. For example, AAV1 and AAV6 are two serotypes that are effective for skeletal muscle transduction (Gao GP et al., Proc Natl Acad Sci USA. 2002; 99: 11854-11859; Xiao W et al., J Virol. 1999; 73: 3994-4003; Chao H et al., Mol Ther. 2000; 2: 619-623).

[0059] Since the development of naturally occurring AAV serotypes into gene therapy vectors, much effort has been focused on understanding the tropism of each serotype so that the virus can be further modified to enhance the efficiency of gene transfer. One approach is to swap domains from one serotype capsid to another, thereby generating hybrid vectors of desired quality from each parent. Since the viral capsid is responsible for cell receptor binding, it is important to understand the viral capsid domains that are critical for binding. Mutagenesis studies on viral capsids (primarily AAV2) before the crystal structure became available were mainly based on functionalization of the capsid surface by adsorption of exogenous moieties, insertion of peptides at random positions, or global mutagenesis at the amino acid level (Choi et al., Curr Gene Ther. 2005 Jun; 5(3): 299-310).

[0060] In some aspects, the present disclosure provides a method for producing rAAV particles having capsid proteins expressed by multiple AAV serotypes. This is achieved by co-infecting production cells with rHSV expression virus and rHSV-rep2capX helper virus, wherein the cap gene product is derived from an AAV serotype other than AAV2 or an AAV serotype other than AAV2. Recombinant AAV vectors have generally been based on the AAV2 capsid. Recently, it has been demonstrated that rAAV vectors based on capsids from AAV1, AAV3, AAV4, AAV5, AAV8, or AAV9 serotypes differ from AAV2 in their tropism.

[0061] Capsids from other AAV serotypes offer advantages over rAAV vectors based on AAV2 capsids in certain in vivo applications. First, the appropriate use of rAAV vectors with specific serotypes can improve the efficiency of in vivo gene delivery to certain target cells that are difficult to infect or not infected at all by AAV2-based vectors. Second, if re-administration of rAAV vectors becomes necessary clinically, it may be advantageous to use rAAV vectors based on other AAV serotypes. It has been shown that re-administration of rAAV vectors with the same capsid may be ineffective, possibly due to the production of neutralizing antibodies produced against the vector. This problem can be avoided by administering rAAV particles whose capsids are composed of proteins from different AAV serotypes and are not affected by the presence of neutralizing antibodies against the first rAAV vector. It will be appreciated that the production of rHSV using the methods described herein allows the construction of recombinant HSV vectors that are similar to rHSV but encode cap genes from other AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV5 to AAV9). In certain aspects, cap genes from different AAVs are used to construct recombinant AAV vectors.

[0062] Example

[0063] Experimental methods

[0064] Generation of a stable CHO pool

[0065] The open reading frames (ORFs) of human HVEM and nectin-1 were downloaded from the NCBI database (GenBank U70321.1 and AF060231.1, respectively). The HVEM and nectin-1 ORFs were codon-optimized for expression in hamster cells using online tools (https: / / www.idtdna.com / CodonOpt and https: / / www.thermofisher.com / us / en / home / life-science / cloning / gene-synthesis / geneart-gene-synthesis / geneoptimizer.html, respectively). Delivery plasmids encoding HVEM or nectin-1 were subcloned into homemade plasmids downstream of an enhanced human cytomegalovirus (CMV) promoter and / or a synthetic promoter (Brown et al., 2017), generating eight different constructs. All constructed plasmids encode glutathione synthetase (GS) under the control of the SV40 promoter, thereby allowing transfected cells to be selected in methionine sulfoximine (Bebbington et al., 1992. Bio / technology (Nature Publishing Company), 10 (2), 169-175). All final plasmids are verified by full plasmid sequencing (Macrogen). Proprietary homemade suspension serum-free adaptive CHO cells are thawed into 125 mL shake flasks and cultured in 30 mL CD-CHO culture medium (Thermo Fisher), which is supplemented with 6 mM L-glutamine (Gibco), dextran sulfate (50 mg / mL, Sigma-Adrich) and incubated at 37°C, 120 rpm stirring in a 6% CO2 humidified incubator. Viable cell density (VCD) and viability are measured every day using a Vi-Cell automated cell counter (Beckman Coulter).

[0066] Stable CHO cell pools were generated according to a standard homemade protocol. Briefly, eight equal aliquots of CHO CAT-S cells (1 × 10 7 The cells were pelleted at 200 × g for 5 minutes (10 viable cells / aliquot). The cell pellets were mixed with 7 μg of each purified, linearized homemade pCLD plasmid and then transfected using the Amaxa Cell Line Nucleofector Kit V (Lonza) according to the manufacturer's instructions. 24 hours after transfection, cell viability was measured, and 75 μM / mL MSX (Sigma-Aldrich) was added to each pool for recombinant cell selection. Aliquots from the recovered cell pools (1 × 10 cells per pool) were tested using FACS staining. 6HVEM and / or Nectin-1 receptor expression in 100 living cells) was detected. Briefly, cells were incubated with 150 μL of 1:200 diluted anti-CD270 (HVEM) eBioscience PE clone eBioHVEM-122 (Invitrogen) and / or 1:200 diluted Nectin-1 monoclonal antibody clone R1.302-PE (Invitrogen) in PBS (Gibco) supplemented with 1% bovine serum albumin (BSA) (Invitrogen) at room temperature in the dark for 15 minutes. After incubation, the diluted antibodies were poured out, and the cells were fixed with Fix and Perm medium A (Life Technologies) and incubated at room temperature in the dark for 15 minutes. The stained cells were then washed twice with PBS and resuspended in FACS buffer (PBS supplemented with 0.1% BSA) for flow cytometry using an LSR II instrument (BD Biosciences). Flow cytometry data were analyzed using FlowJo v10.0 software (Tree Star, Inc).

[0067] rHSV-1 infection in stable CHO cells

[0068] Aliquots (1×10 cells) from each recovered cell pool and CHO CAT-S host cells were infected with rHSV-GFP (MOI=10) in 6-well cell culture plates (Corning) in a humidified 5% CO2 incubator at 37°C. 6 The infected pools were centrifuged at 1200 rpm for 5 minutes and the excess viral supernatant was discarded. The infected cell pellets were resuspended in CD-CHO culture medium and incubated at 37°C in a 5% CO2 humidified incubator connected to an IncuCyte (Sartorius). Cell imaging of GFP expression from the infected pools was determined using the IncuCyte default settings, and GFP expression was captured from each infected cell pool every 12 hours for a total of five time points.

[0069] rAAV9-GFP vector production in stable CHO cells

[0070] Aliquots (1 × 10 610 viable cells) were infected with an MOI of 1:1 from rHSV-AAV9 rep / cap:rHSV-AAV-GFP vector in 6-well culture plates (Corning). The infected cell pools were incubated in a 5% CO2 humidified incubator at 37°C for 24 hours. After 24 hours, the infected cell supernatants were collected and the rAAV9-GFP titer was tested using qPCR. Briefly, the collected supernatants were digested with DNase I (200 U / μL, Invitrogen) at 37°C for 1 hour and then incubated at 95°C for 10 minutes to inactivate the enzyme. The digested samples were then incubated with an equal volume of proteinase K digestion mix (200 mM NaCl, 20 mM Tris-HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid, pH 8.0; 0.5% sodium dodecyl sulfate and proteinase K (20 mg / mL) at 55°C for 1 hour, followed by enzyme inactivation at 95°C for 10 minutes. Absolute qPCR quantification reactions were performed using a PCR thermal cycler (QuantaBioQ, Qiagen) in 20 μL reactions containing TaqMan Fast Universal PCR 2x Master Mix (Applied Biosystems) in addition to 5 μL of diluted template using homemade standard linearized AAV plasmids. Biosystems) and 20 μM of each CMV-forward primer (5'-TTCCTACTTGGCAGTACATCTACG'-3'), CMV-reverse primer (5'-GTCAATGGGGTGGAGACTTGG-'3'), and CMV probe (5'-FAM-TGAGTCAAACCGCTATCCACGCCCA-NFQ-'3'). The PCR cycling profile was 95°C for 2 minutes and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

[0071] Generation and testing of rHSV-GFP infection in CHO-HVEM expression clones

[0072] Single cell sedimentation cloning was performed using a BD Influx cell sorter (BD Biosciences) according to Evans et al., 2015 (Biotechnology Progress, 31(5), 1172-1178). Briefly, 3 × 10 6An aliquot of live HVEM CHO expressing cells was stained with anti-human CD270 (HVEM)-PE (Invitrogen) antibody diluted 1:200 in PBS at room temperature in the dark for 15 minutes. The stained cells were washed twice with sterile PBS, pelleted at 200 × g for 5 minutes, and then resuspended in 1 mL of sorting buffer. Cells were sorted from the PE gated fraction into two 384-well plates (Agilent) containing homemade conditioned medium supplemented with 50 μM MSX / mL. The selected recovered clones were further tested for rHSV-GFP infection, and the average GFP expression was calculated from two time points (24 and 48) hpi using IncuCyte as described above.

[0073] Production of rAAV6.2-GFP vector in final CHO-HVEM clones

[0074] The selected clones were also further tested for rAAV6.2-GFP vector production using an MOI of 1:1 for rHSV-AAV6.2rep / cap:rHSV-GFP. All infected clones were incubated in a 5% CO2 humidified incubator at 37°C with agitation at 120 rpm for three days. Aliquots (1 mL) were harvested from each co-infected clone 24 hours and 48 hours after co-infection and centrifuged at 200×g for 5 minutes. The harvested samples (cell pellets) were then prepared for rAAV titration using qPCR. Briefly, the infected cell pellets were collected, mixed with AAV lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl), and subjected to three freeze / thaw cycles in an isopropanol dry ice bath, followed by centrifugation at 12,000 rpm for 30 minutes at 4°C. After centrifugation, the supernatant was collected and the rAAV6.2-GFP titer was determined using qPCR as described previously. In subsequent experiments, two of the final selected clones were further tested for rAAV6.2-GFP vector production using different MOIs and incubation temperatures. As described previously, rAAV6.2-GFP titers were determined from harvested cell pellets 24 hours after co-infection and titrated using qPCR. Purification of rAAV produced in the final selected CHO clones was completed according to (Negrini et al. CurT Protoc Neurosci. 2020 Sep;93(1):e103). rAAV produced in the final selected CHO clones was purified using the polyethylene glycol (PEG) chloroform method according to Negrini et al., with some modifications (Negrini et al., 2020), including cell lysis and rHSV-1 inactivation using 0.5% Triton X-100 (v / v), followed by precipitation using 8% polyethylene glycol 8000 and 150 mM NaCl. The viral pellet was further treated with Benzonase (Sigma; 50 U / mL) and RNase (Invitrogen; 10 μg / mL) at 37°C for 1 hour, followed by treatment with 1:1 chloroform (Sigma). The aqueous layer after chloroform treatment was collected and concentrated using an AMICON filter (Sigma). The concentrated virus was stored at -80°C until use.

[0075] Analytical characterization of CHO-derived rAAV

[0076] Western blotting was used to test the capsid protein expression of purified rAAV. Briefly, by adding an appropriate volume of 4 × NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Life Technologies) and 10 × NuPAGE sample reducing agent (Thermo Fisher), the purified rAAV6.2-GFP and rAAV9-GFP vectors produced in the final selected clones using rHSV-1 vectors of different MOI were prepared for SDS-PAGE gel. The samples were then incubated at 70 ° C for 10 minutes. For each serotype, an equal volume of rAAV was loaded onto a Bolt 4-12% Bis-Tris plus 12-well gel (Invitrogen) and run using 1 × NuPAGE running buffer (Thermo Fisher). After the run, the gel was dry-transferred using iBolt 2NC ministack (Invitrogen) and then blocked for 1 hour at room temperature in 5% skim milk (Amresco) diluted with TBS (Bio-Rad). After blocking, the membrane was incubated overnight at 4 ° C with gentle shaking with 1: 200AAVVP1, VP2, VP35% skim milk diluted antibodies (Genentech, USA). After incubation, the blot was washed three times with TBS (Life Technologies) supplemented with 0.1% Tween, then incubated with 1: 100 goat anti-mouse IgG (Thermo Scientific) for 1 hour, and then washed. The membrane was incubated with Supersignal West Pico Plus substrate (Thermo Scientific) and then imaged using Amersham Imager 680 (GE Healthcare). In subsequent experiments, purified rAAV from the final selected CHO clone was visualized using a miniature transmission electron microscope (Mini-TEM; Vironova). In brief, PEG-chloroform-purified rAAV6.2-GFP and rAAV9-GFP samples were placed on 400-mesh glow-discharge carbon grids by first inverting the grid and placing it above the top of a 10 μL rAAV droplet deposited on paraffin film for 30 seconds. Excess sample was removed by gently touching the edge of the grid to a Whatman filter paper. The grid was then washed twice with two 20 μL drops of double-distilled water. The grid containing the sample was then stained with a 20 μL drop of 1.5% uranyl acetate for 10 seconds. Excess stain was removed by gently touching the edge of the grid to a Whatman filter paper. The rAAV sample was then visualized using a Mini-TEM instrument.In a separate experiment, rAAV capsid protein (VP1:VP2:VP3) ratio analysis was performed using a self-developed capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method according to Kurasawa et al., (Mol Ther Methods Clin Dev. 2020 Oct 4;19:330-340).

[0077] Infectious rHSV-1 residues from purified CHO-derived rAAV

[0078] V27 cells expressing a stable copy of rHSV-1 ICP27 protein were cultured at 0.5 × 10 6 Live cells / well were seeded in 6-well culture plates and grown overnight in DMEM supplemented with 10% FBS (Gibco) and 500 μg / mL Geneticin (Gibco). After 16 hours, the cells were washed twice with sterile PBS and then inoculated with purified rAAV6.2-GFP and rAAV9-GFP vectors at a dilution of 1:100 (~10 10 vg / mL) infection. rHSV-AAV-GFP vector was used as a positive control (MOI = 0.15 PFU / cell). Infected cells were incubated at 37°C for 2 hours for virus adsorption. After incubation, excess virus was removed and infection medium (DMEM supplemented with 2% FBS) was added. The plates were incubated with IncuCyte and monitored for 4 days to capture any cytopathic effects.

[0079] Infectivity and in vitro transduction of CHO-derived rAAV

[0080] For infectivity, Ad293 cells were seeded in 96-well culture plates (2×104 cells / well) and incubated overnight in a humidified 5% CO2 incubator at 37°C. Ten-fold dilutions of rAAV6.2-GFP, rAAV9-GFP, and / or rAAV9-ZsGreen produced in CHO clones and purified with PEG-chloroform and / or produced in HEK293 cells using triple transfection (Kimura et al., Sci Rep 9, 13601, 2019) and purified by affinity chromatography were performed. Each virus dilution was used to infect four wells, and the infected cells were incubated in a 37°C, 5% CO2 incubator for 5 days. On day 5 post-infection, infected cells were imaged for GFP / Zs-Green expression using the IncuCyte default settings, and viral titers were calculated using the method of Reed and Muench (Reed and Muench, American Journal of Epidemiology; 27; 3; 493-497, 1938). For transduction analysis, Ad293 cells were cultured as described above and then infected with different multiplicities of transduction (MOT) of rAAV from CHO origin and / or similar vectors produced in HEK293 cells using triple transfection as described above.

[0081] Biodistribution of CHO-derived rAAV

[0082] All animal experiments were approved by the Institutional Animal Care and Use Committee of AstraZeneca (Gaithersburg, MD, USA). Eight-week-old male C57bl / 6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were divided into five groups (n = 5 per group) and all mice were inoculated with 1 × 10 insulin via the tail vein using a BD insulin syringe (Becton Dickinson). 11vg / 100 μL of appropriate rAAV vector (or saline). Vaccinated mice were monitored daily for any clinical signs of disease. Three weeks after injection, mice were euthanized by CO2 and organs were harvested. Half of the harvested tissue was sliced and frozen in dry ice in a microcentrifuge tube for qPCR analysis, and the other half was fixed in 10% neutral buffered formalin for histological work. DNA was extracted from the harvested tissue using the All-Prep DNA / RNAMini kit (Qiagen) according to the manufacturer's instructions. qPCR reactions were performed on a QuantStudio 7Flex using homemade linearized plasmids: pAAV-GFP was used for Groups 1, 2, and 3, and pAAV-ZsGreen was used for Group 4. Extracted genomic DNA (100 ng) was used as a template, using specific GFP primers and probes (forward 5'-GAACCGCATCGAGCTGAA-'3, reverse 5'-TGCTTGTCGGCCATGATATAG-'3 and probe 5' / 56-FAM / ATCGACTTC / ZEN / AAGGAGGACGGCAAC / 3IABkFQ'3 and ZsGreen primers and probe forward 5'-GTACCACGAGTCCAAGTTCTAC-'3, reverse 5'-CACGTCGCCCTTCAAGAT-'3 and probe 5' / 56-FAM / CCCGTGATG / ZEN / AAGAAGATGACCGACAA / 3IABkFQ / '3). Cycling conditions included an initial denaturation at 95°C for 3 seconds, 40 cycles at 95°C for 10 seconds, and an annealing / extension at 60°C for 20 seconds. For histology, thin liver tissue sections from collected tissue were prepared using a Leica 3050s microtome (Germany), stained, mounted, and assessed for GFP detection using confocal microscopy. GFP quantification from liver slides was done using an axioscanner.

[0083] Engineered rHSV-1 vector production cell pool

[0084] Two proprietary suspension cell lines were selected; based on CHO-K1 and BHK-21, for engineering work. For suspension CHO cells, two strategies were used: (1) random integration, in which the HSV-1 ICP27 ORF (GenBank AB235845.1) sequence was codon-optimized for hamster cell expression by IDT, chemically synthesized, and then subcloned downstream of the CMV promoter into a homemade plasmid encoding a puromycin cassette for selection. For site integration, exon 1 of the C12 Orf35 locus from the CHO genome (GenBank XM027430029) was selected as one of the transcriptional hotspots (Zhao et al., Appl Microbiol Biotechnol. 2018 Jul; 102(14): 6105-6117). sgRNA target sequences were selected using the CRISPy bioinformatics tool with default parameters according to (Ronda et al., 2014). According to the manufacturer's instructions, the GeneArt CRISPR CD4 kit (Invitrogen) was used to synthesize the selected gRNA target (5'GGACTTAACCACTCGATGGC-'3) by IDT and delivered as gblocks, annealed, and subcloned into a linearized CRISPR nuclease expression vector (GeneArt CRISPRCD4) backbone to produce an sgRNA expression vector. The donor DNA plasmid was constructed using the backbone of a homemade plasmid to avoid any protospacer sequence adjacent to the motif (PAM) site identical to the gRNA target. The 5' and 3' homology arms (750 base pairs per arm) flanking the sgRNA target sequence were chemically synthesized (IDT), containing 110 nucleotides as gene linkers, containing different restriction sites for cloning. The final AAV CHO clone was transfected according to the process described above. 5 μg puromycin / mL was used to select the transfected cell pool for two weeks. Based on the method described previously (Evans et al., 2015), clones were generated by single-cell sedimentation in 384-well plates using a BD Influx cell sorter (BD Biosciences) using the anti-HU CD270 (HVEM) eBioscience PE clone eBioHVEM-122. (2) For BHK-21 suspension cells, homemade serum-free suspension-adapted BHK-21 cells were appropriately maintained in Xell HEK TF medium (Xell AG, Germany). Cells were seeded at a density of 0.3 × 106 cells / ml in 30 ml of medium (Nunc, Denmark) in a 125 ml shake flask and incubated in a shaking incubator at 120 rpm and 5% CO2 for three days.ICP27 (ORF) from the HSV-1 genome (GenBank KM222723.1) was downloaded and codon optimized, synthesized, and delivered in a commercial plasmid using the online IDT tool (https: / / www.idtdna.com / CodonOpt). The codon-optimized ICP27 gene was subcloned into a homemade plasmid downstream of the CMV promoter or the endogenous ICP27 promoter, and restriction enzyme cloning was used to generate pCLD-CMV-ICP27 and pCLD-EN-ICP27, respectively. The pCLD plasmid constructed above contains a puromycin cassette for selection. Another form of ICP27 (uncodon-optimized) ORF was synthesized by GeneArt (Thermo Fisher, USA) and subcloned into a commercial expression plasmid downstream of the CMV promoter using BamHI-NotI restriction cloning to generate a third plasmid (called pCDNA-ICP27). pCDNA-ICP27 contains a neomycin cassette for selection. Three plasmids were amplified in competent DH5α and purified using maxiprep (Qiagen, USA). Final plasmid was confirmed by sanger sequencing (Macrogen, USA). According to the manufacturer's instructions, Amaxa nuclear transfection kit L (Lonza, USA) was used to transfect aliquots (each aliquot 7x106 cells) of BHK-21 cells with linearized plasmid (each 2.5 μg) in nucleofector II. Forty-eight hours after transfection, the transfected cells were placed under 400 μg / ml geneticin (Gibco, USA) and / or 10 μg puromycin (Gibco, USA) selection conditions for three weeks.

[0085] rHSV-1 infection in selected CHO-ICP27 clones

[0086] The final selected clones were tested for rHSV-1 infection. Briefly, aliquots (1×10 6viable cells) and then incubated on ice for 20 minutes and then at 37°C for 2 hours. After incubation, excess virus was decanted and the infected cells were washed twice with sterile PBS to remove any residual rHSV-AAV9 vector. After washing, the infected cells were covered with homemade culture medium (2 mL / well) in a 6-well plate and incubated in a 5% CO2 humidified incubator at 37°C for 24 hours. After 24 hours, 1 mL of supernatant from the infected cells was collected and inoculated on V27 cells to determine the cytopathic effect. The virus harvested from the inoculated V27 cells was purified by ultracentrifugation and detected by Western blotting using an anti-HSV-1 glycoprotein D antibody (EMD Millipore Corp). In separate experiments, the final CHO clone and / or V27 cells were infected with different MOIs of rHSV-AAV9 using the same method described above. The infected cells were then incubated in 6-well plates containing homemade culture medium (for CHO) or DMEM-2% FBS for V27 in a humidified incubator at 33° C. with 5% CO 2 for two days. Clarified virus from the lysed cultures was harvested and titrated on V27 cells by plaque assay (Kang et al., 2009. Gene Therapy 16, 229-239).

[0087] Production of rHSV-1 in BHK-21-ICP27 cells

[0088] The recovered BHK-ICP27 pool was tested for rHSV-1 yield. Briefly, cells were infected with rHSV-AAV6.2 vector (MOI = 0.15 PFU) by direct inoculation in XellHEK TF medium supplemented with 4% FBS or under serum-free conditions. The infected cells were incubated in a 37°C, 120 rpm, 5% CO2 humidified incubator for 3 to 4 days. Cell viability and density were measured daily using Vi-Cell after infection. After 3-4 days, the infected culture was subjected to three freeze-thaw cycles in an isopropanol dry ice bath and then centrifuged at 4500 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was ultracentrifuged at 10,000 rpm for 75 minutes at 4°C using a JA 20 rotor. The resuspended virus was titrated on V27 cells using a plaque assay according to (Kang et al., 2009).

[0089] Statistical analysis

[0090] One-way ANOVA with Tukey-Kramer post hoc was used to compare GFP expression after rHSV-1 infection in CHO cell pools and to compare rAAV9-GFP titers produced from different cell pools. Two-way ANOVA was used to compare rAAV6.2-GFP titers produced from different clones at two different time points. The infectivity titers of rAAV6.2-GFP and rAAV9-GFP produced in CHO or rAAV6.2-GFP and rAAV9-ZsGreen produced by triple transient transfection were compared using a two-tailed Mann-Whitney test. RAAV6.2-GFP, rAAV9-GFP and / or rAAV9-ZsGreen titers in harvested mouse tissues were compared using a Kruskal-Wallis test and a Dunn's test for correction. GraphPad Prism version 9.1.2 (GraphPad Software Inc) was used for all tests, and p values ≤ 0.05 were considered significant differences.

[0091] result

[0092] Generation of a stable CHO pool

[0093] Eight different vectors using homemade plasmid backbones were successfully constructed: (1) two vectors encoding the codon-optimized or non-codon-optimized HVEM ORF flanked by a CMV promoter and SV40 poly(A); (2) two vectors encoding the codon-optimized or non-codon-optimized Nectin-1 ORF flanked by a CMV promoter and BGH poly(A); (3) two vectors encoding the codon-optimized or non-codon-optimized HVEM and Nectin-1 ORFs flanked by a CMV promoter and SV40 poly(A), respectively; and (4) two vectors encoding the codon-optimized or non-codon-optimized HVEM and Nectin-1 ORFs flanked by a synthetic promoter (Spro), SV40 poly(A), and BGH poly(A), respectively (Figures 1a and b). Ten days after MSX selection, aliquots of each recovered stable cell pool (1×10 6 viable cells / pool) were tested for HVEM and / or Nectin-1 receptor expression using FACS staining. Pools 1 (CMV-HVEM), 2 (CMV-HVEM-Co), 5 (CMV-HVEM-Nectin-1), 6 (CMV-HVEM-Co-Nectin-1-Co), 7 (Spro-HVEM-Nectin-1), and 8 (Spro-HVEM-Co-Nectin-1-Co) showed 32.35%, 65%, 60.9%, 51%, 60%, and 53.8% of HVEM-expressing cells, respectively ( FIG1 c ), while Pool 3 (CMV-HVEM-Co) showed 32.35%, 65%, 60.9%, 51%, 60%, and 53.8% of HVEM-expressing cells, respectively ( FIG1 c ). -Nectin-1), Pool 4 (CMV-Nectin-1-Co), Pool 5 (CMV-HVEM-Nectin-1), Pool 6 (CMV-HVEM-Co-Nectin-1-Co), Pool 7 (Spro-HVEM-Nectin-1), and Pool 8 (Spro-HVEM-Co-Nectin-1-Co) showed 69.75%, 53.45%, 61.3%, 49.3%, 63.7%, and 53.8% of Nectin-1 expressing cells, respectively ( Figure 1d Interestingly, dual pools (i.e., pools 5, 6, 7, and 8) showed good expression of HVEM and Nectin-1, indicating that the CMV and synthetic promoters were comparable in driving high-level expression of HVEM and Nectin-1 proteins. Furthermore, no significant differences in HVEM and Nectin-1 receptor expression were observed using non-codon-optimized and / or codon-optimized protein forms.

[0094] rHSV-1 infection and rAAV9-GFP production in stable CHO cells

[0095] Since these receptors are stably expressed in these CHO pools, it is crucial to understand which construct provides the best rHSV-1 infection and subsequent rAAV expression after co-infection with rHSV-1 vectors. Each stable pool generated above was infected with rHSV-GFP at MOI=10, with the infected host CHO cells serving as negative controls. GFP expression data were collected from all infected stable cell pools (n=8) every 12 hours to a total of 60 hours after infection. Compared to the infected host CHO cells showing the lowest GFP expression, all eight infected cell pools began to show significant average GFP expression (Fig. 2a) 12 hours after infection (hpi). At 12hpi, the highest level of GFP expression was observed only in the HVEM expression pool, with pools 1 and 2 showing the highest GFP expression (p value < 0.0001 compared to the infected host CHO cells), followed by pools 4 and 3 (p value < 0.01 for codon optimization compared to the infected host CHO cells). Furthermore, stable cell pools 1 and 2 showed the highest overall GFP expression, with pool 1 outperforming pool 2 in terms of overall mean GFP expression.

[0096] One day after co-infection, cell supernatants were collected and rAAV9-GFP vectors were titrated using qPCR. Compared with other pool numbers, pool 1 showed the highest rAAV9-GFP physical titer, with an average of 8.2 log10 vg / mL; pools 2, 3, 4, 5, 6, 7, and 8 showed average titers of 7.97, 7.76, 7.99, 8.07, 7.68, 7.71, and 7.99 log10 vg / mL, respectively. 10 vg / mL. However, the difference in qPCR titers of rAAV9-GFP vectors produced in pool 1 was not significant compared with the titers produced in the other pools. Compared with the rAAV9-GFP titers obtained from infected host CHO cells (6.84 log 10 vg / mL), the rAAV9-GFP titers generated from all pools were significant, with p < 0.0001 (Figure 2b). These data indicate that pool 1 showed the highest average GFP expression and the highest rAAV9-GFP titer after rHSV-GFP vector infection, and therefore this pool was selected for single-cell cloning.

[0097] Generation and testing of CHO-HVEM expression clones for rHSV-GFP infection

[0098] Selection of individual high and medium HVEM expressing CHO clones ( Figure 3) and deposited them into two 384-well plates using homemade conditioned medium for two weeks. The deposition of single cells / well was verified by imaging using Cellavista (Evans et al., 2015). Sixty-four clones were recovered after two weeks, showing high viability (90-95%) and good growth curves. The selected clones were further passaged three times in 96-deep-well plates containing homemade medium supplemented with MSX. After three passages, twenty-four of the initial 64 clones showed good HVEM expression by FACS staining (Figure 4a). These clones were expanded and further tested for rHSV1-GFP vector infection. The average GFP expression was calculated from two time points (24 and 48) hpi. No statistically significant differences in the average GPF expression levels were detected among the tested clones (Figure 4b).

[0099] rAAV production in CHO-HVEM expression clones

[0100] Eight clones (designated CHO-HV-C1, CHO-HV-C13, CHO-HV-C15, CHO-HV-C23, CHO-HV-C24, CHO-HV-C46, CHO-HV-C62, and CHO-HV-C64) that showed the highest average GFP expression after infection with the rHSV-GFP vector were selected to evaluate their ability to produce rAAV by co-infection with two rHSV-1 vectors, one containing the AAV2 rep and AAV6.2 cap genes and the other containing the GFP gene, at an MOI of 1:1. After infection, the viability of the eight clones decreased rapidly within a few days, compared to only a slight decrease (3-7%) in the infected host CHO cells during the co-infection process when incubated at 37°C (Figure 5a). Thus, compared with the slight decrease in the infected host CHO cells (0.3×10 6 The viable cell density (VCD) of all co-infected clones also showed a significant decrease compared to the control group (Figure 5b). As shown by the sharp decrease in cell viability and VCD compared to the host CHO cells, it seems that the AAV rep protein has a deleterious effect on the metabolism of the infected engineered CHO cells, wherein the rHSV-1 vector undergoes degradation after cell invasion when the co-infected cells are incubated at 37°C. Therefore, it is envisaged to minimize this deleterious effect by lowering the incubation temperature.

[0101] Interestingly, clone #1 (designated CHO-HV-C1) produced the highest rAAV6.2-GFP vector titer per 1 mL of cell lysate (~8.83 log 10 vg / mL; 1×10 6 cells) at 24 hpi, compared to 7.74 to 8.34 log 10vg / mL. However, the rAAV6.2-GFP titers produced in the CHO-HV-C1 clone were not significantly different from those produced by the other clones tested (p=0.89). However, they were all significantly higher than 6.24 log at 24 hpi. 10 vg / mL of co-infected host CHO cells. (Figure 5c). The rAAV6.2-GFP titers from all eight co-infected clones decreased slightly at 48 hpi (Figure 5c).

[0102] Clones CHO-HV-C1 and CHO-HV-C62 produced the highest titers of rAAV6.2-GFP vectors. Clone CHO-HV-C1 is a high HVEM expression clone, while the CHO-HV-C62 clone is a medium HVEM expression clone. In subsequent experiments, the final selected clones (CHO-HV-C1 and CHO-HV-C62) were tested for rAAV6.2-GFP vector production using MOIs of 2:1, 3:1, and 4:1 for rHSV-AAV6.2 and rHSV-GFP, respectively.

[0103] Interestingly, compared with the titers obtained using MOI 1:1, MOIs 2:1 and 3:1 showed a significant increase in titers per 10 at 24 hpi. 6 The rAAV6.2-GFP titer of the cell lysate did not show a significant improvement (data not shown). On the other hand, MOI 4:1 significantly improved the rAAV6.2-GFP physical titer produced in both CHO-HV-C1 and CHO-HV-C62 clones (p = 0.0211). In addition, the CHO-HV-C1 clone outperformed CHO-HV-C62 in producing rAAV6.2-GFP vector at MOI 4:1 and produced 100 ng / mL of rAAV6.2-GFP vector at 24 hpi. 6 The cell lysates produced 9.89 and 9.37 log 10 vg / mL (p=0.0261) (Fig. 5d), indicating that the MOI of rHSV-1 has a significant effect on rAAV6.2-GFP in CHO cells.

[0104] Since CHO-HV-C1 was the most productive clone for rAAV6.2-GFP production, its ability to produce other AAV serotypes 8 and 9 expressing the GFP transgene was further tested. Using the same optimal infection parameters as described in the previous experiment, 1 × 10 of rAAV8-GFP and rAAV9-GFP vectors were generated at 24 hpi. 6 9.21 and 9.4 log cells 10The average titer of vg / mL was 24 hpi. Harvest at 30 hpi showed that the titer of rAAV8-GFP and rAAV9-GFP vectors decreased to 8.36 and 8.98 log10 vg / mL, respectively (Figure 5e). These data indicate that the above infection parameters work across the serotypes tested. In addition, cell lysates produced the highest physical titers of the AAV serotypes tested at 24 hpi compared to the titers at 30 hpi and 48 hpi.

[0105] The cell viability of CHO-HV-C1 cells co-infected for the production of rAAV8-GFP and / or rAAV9-GFP vectors decreased sharply at 24 hpi, similar to the case of rAAV6.2-GFP vector production, indicating that the decrease in cell viability is not AAV serotype specific. The sharp decrease in cell viability after rHSV-1 vector co-infection may affect the final rAAV titer, so the effect of a temperature shift from 37°C to 33°C was tested, which showed a significant improvement in cell viability and rAAV6.2-GFP titer after co-infection ( Figure 6 ). Therefore, coinfection with an MOI of 4:1 (rHSV-1AAV6.2:rHSV-1-GFP) at 33°C was tested. Compared to cultures incubated at 37°C, cell viability of coinfected cultures at 24 hpi was only slightly decreased (5-7%), rAAV6.2-GFP titers in lysates were slightly improved, and titers in culture medium increased approximately twofold (data not shown). This finding suggests that incubation temperature after coinfection is an essential factor for cell viability and / or rAAV production in the CHO platform.

[0106] Analytical characterization of CHO-derived rAAV

[0107] The entire process of harvesting and purifying rAAV vectors takes one day to perform the self-developed PEG-chloroform method ( Figure 7). Briefly, all infected cultures were lysed with 0.5% Triton X-100 (v / v) under gentle shaking conditions for 1 to 3 hours. The Triton-treated culture was then centrifuged at 1200 rpm for 5 minutes, and the supernatant was subsequently filtered at 0.2 μm using a PES filter. The filtrate was mixed with 1 / 4 volume of 40% PEG8000 / 5M NaCl on ice for 1 hour, and then centrifuged at 4500 rpm for 40 minutes at 4°C. The PEG virus pellet was resuspended in resuspension buffer and treated with Benzonase (50 U / ml) and RNase A (20 μg / ml) at 37°C for 1 hour, with tube mixing every 15 minutes. After Benzonase treatment, the mixture was mixed with chloroform in a 1:1 ratio and centrifuged at 12000 rpm for 5 minutes. After centrifugation, the chloroform was evaporated under a biosafety cabinet, the aqueous layer was collected, and then concentrated. The final purified rAAV was stored at -80°C.

[0108] Western blots of equal volumes of purified rAAV produced in the CHO-HV-C1 clone using different MOIs of HSV-1 showed comparable expression of VP1, VP2, and VP3 capsid proteins (Figure 8a). In addition, examination of purified rAAV6.2-GFP and rAAV9-GFP vectors using mini-TEM showed 91% and 79.5% intact capsids for rAAV6.2-GFP and rAAV9-GFP, respectively (Figures 8b and 8c). In addition, analysis of the VP1:VP2:VP3 molar ratios of these rAAVs was consistent with those reported in the literature (Figure 8d). These data show that rAAV vectors produced in CHO cells have good expression of capsid proteins, with a high percentage of AAV complete capsids. However, the high percentage of complete capsids obtained may be related to the purification method used, so it is worthwhile to re-investigate the percentage of complete capsids after using other purification methods (e.g., chromatography).

[0109] Residual infectious HSV-1 from CHO-derived rAAV

[0110] rHSV-GFP vector (MOI 0.15 PFU / cell) was used as a positive control. 10Purified rAAV6.2-GFP and rAAV9-GFP vectors were inoculated on an HSV-1 complementing cell line (V27) at 500 μg / mL of each vector to examine any residual infectious HSV-1 vector in the purified drug substance. No cytopathic effect was observed in the wells inoculated with purified rAAV6.2-GFP or rAAV9-GFP vectors on day 4 post-infection, whereas typical cytopathic effect was observed in the wells inoculated with rHSV-GFP vector starting on day 2 post-infection in the form of rounded infected cells and detachment of cell membrane sheets, leading to complete detachment of cell membrane sheets on day 3 post-infection ( Figure 9 These data indicate that the purification method developed by ourselves is highly efficient in inactivating rHSV-1 vectors, and no residual infectious rHSV-1 was detected in the purified rAAV.

[0111] Infectivity and in vitro transduction of CHO-derived rAAV

[0112] The infectivity and in vitro transduction efficacy of rAAV produced in CHO cells and purified using PEG-chloroform were tested to compare them with those of rAAV produced using a standard triple transient transfection method and purified by affinity chromatography. For infectivity, GFP expression from infected wells was recorded at day 5 post-infection using the default settings of the IncuCyte. rAAV6.2-GFP vectors produced in the CHO-HV-C1 clone (referred to as rAAV6.2-CHO) and rAAV6.2-GFP produced in HEK293 cells (referred to as rAAV6.2-GFP TTT) showed a similar expression to 1.65 × 10 7 and 1.1×10 7 Tissue culture infectious dose 50 (TCID 50 On the other hand, the rAAV9-GFP vector produced in the CHO-HV-C1 clone (referred to as rAAV9-GFPCHO) showed an infectious titer of 6×10 6 TCID 50 / mL, in contrast, rAAV9-ZsGreen vector produced in HEK293 cells using triple transient transfection (referred to as rAAV9-Zs-Green-TTT) showed 6×10 5 TCID 50 / mL (Figure 10b).

[0113] Different multiplicities of transfection (MOT) from the four vector preparations described above were tested in Ad293 cells, ranging from 2 × 10 5 As low as 1×10 3vg / cell for comparison of transduction, and average GFP expression was recorded on day 3 post-infection using the IncuCyte default settings. rAAV6.2-GFP-CHO and rAAV6.2-GFP-TTT showed efficient transduction at all MOTs tested. On the other hand, rAAV9-GFP-CHO showed higher transduction efficacy (Figure 10c) compared to rAAV9-ZsGreen-TTT, consistent with the observed infectivity data. These data indicate that rAAV6.2-GFP-CHO and rAAV9-GFP-CHO have good in vitro infectivity and transduction activity. In addition, the higher infectivity and transduction of rAAV9-GFP-CHO compared to rAAV9-Zs-Green-TTT observed herein may be related to the differences in the percentage of complete capsid, purification method, and formulation buffer used for each of the different sample preparations.

[0114] Biodistribution of CHO-derived AAV

[0115] The biodistribution of rAAV6.2-GFP-CHO and rAAV9-GFP-CHO was evaluated in parallel with rAAV6.2-GFP-TTT and rAAV9-Zs-Green-TTT to determine whether the in vivo behavior mimicked the in vitro data. Twenty-five three-week-old mice were divided into five groups (n=5 per group). Mice were inoculated with rAAV6.2-GFP-CHO (G1), rAAV9-GFP-CHO (G2), rAAV6.2-GFP-TTT (G3), rAAV9-Zs-Green-TTT (G4), or PBS (G5). All mice were inoculated intravenously in the tail vein for 10 min according to their grouping. 11 vg rAAV or 100 μL PBS. Three weeks after vaccination, the vaccinated mice were euthanized and tissues with high tropism for these tissues (heart, liver, lung, kidney and skeletal muscle) were harvested from each vaccinated animal (Figure 11a). The rAAV titer in the homogenized tissues of these tissues was assessed using qPCR (targeting GFP and / or Zs-Green genes), and histopathological examination was performed using confocal microscopy. For qPCR, mice from G1 showed lower GFP copy numbers than mice from G3 in all harvested tissues except the kidney, where mice from G1 showed a GFP copy number of ∼3.89 × 10 4 AAV genomes / mg DNA has a higher average GFP copy number / mg, ~5.38×10 4AAV genomes / mg DNA (Figure 11b), although these differences were not statistically significant. In addition, the rAAV6.2-GFP copy number from the liver of G1 and G3 showed the highest GFP copies among all vaccinated mice compared to the titers of other tissues from both groups. Interestingly, mice in G2 showed a higher average GFP copy number / mg DNA in the heart, lung, kidney, and skeletal muscle than mice in G4. However, the liver from G4 showed a higher average GFP copy number / mg DNA than the liver from G2, and the average titer was 3.79×10 6 and 2.11×10 6 In addition, the GFP titers from the liver of G2 and / or G4 showed the highest GFP copies among all vaccinated mice from both groups compared with the titers from other tissues ( FIG11c ).

[0116] Thin liver tissue sections from five groups were prepared and GFP expression was examined using confocal microscopy. As expected from the qPCR data, clear GFP and / or Zs-Green signals were observed in the livers of all inoculation groups except G5 (Figure 11d). In addition, the quantification of GFP and Zs-Green from liver slides using a slide scanner with a self-developed script showed that liver sections from G3 showed significant biodistribution compared to liver sections from G1, which was correlated with the qPCR data. In addition, liver sections from G4 also showed a higher Zs-Green signal than the GFP signal from G2; however, the difference was not significant. No GFP signal was detected in the mock-infected group (G5) inoculated with sterile PBS. These data indicate that rAAV produced in CHO cells shows good in vivo transduction after tail vein injection. In addition, the lower GFP in liver sections, especially the GFP from G1, may be related to several factors, such as the presence of impurities that cannot be completely eliminated with the PEG-chloroform method.

[0117] Engineering CHO-HV-C1 cells for rHSV-1 production

[0118] For random integration, a self-constructed linearized plasmid containing a synthetic Chinese hamster codon-optimized HSV-1 ICP27 ORF downstream of the CMV promoter and upstream of the SV40 polyadenylation, and a puromycin ORF downstream of the CMV promoter and BGH polyadenylation was used for transfection ( FIG. 12 a ).

[0119] For site integration, two plasmids were constructed, the first containing a codon-optimized ICP27 ORF downstream of the CMV promoter and upstream of SV40 polyadenylation, followed by a puromycin cassette flanked by the SV40 promoter and SV40 polyadenylation. The total length of the two cassettes was 4.1 kb, flanked by right and left homology arms (750 bp each) (Figure 12b). The second plasmid contained a synthetic sgRNA for CHO exon 1C12orf35. After 3 weeks of double selection using 5 μg / mL puromycin and 50 μM MSX / mL, pools from random integration and / or site integration were recovered. After three passages and double selection in homemade culture medium, twenty-four clones were selected, including seven site-edited clones (clones 1-7) and 17 random integration clones showing high growth viability and ICP27 expression (Figure 12c). The random integration clone #11 (designated CHO-HV-ICP27-C11), which showed the highest growth curve and ICP27 expression, was selected for further testing for rHSV-1 vector production.

[0120] rHSV-1 vector infection and production in CHO-HV-ICP27-C11 cells

[0121] The CHO-HV-ICP27-C11 clone was infected with rHSV-AAV9 (MOI=10) and incubated at 37°C for 2 hours for virus adsorption. After 2 hours, the infected cells were washed twice with sterile 1×PBS to remove any viral residues. The infected cells were then incubated in a 5% CO2 static humidified incubator at 37°C for 24 hours. On the second day, 1 mL of clarified supernatant from the infected cells was passaged on V27 cells. Two days after infection, the cells became rounded and the appearance of infected cell membrane detachment appeared. In addition, after infection with rHSV-1 virus propagated in the CHO-HV-ICP27-C11 clone, expression of HSV-1 glycoprotein D (gD) was observed in the V27 cell lysate (data not shown). This result indicates that the CHO-HV-ICP27-C11 clone supports productive infection of the rHSV-1 vector.

[0122] Therefore, the production capacity of the CHO-HV-ICP27-C11 clone of V27 cells was compared. CHO-HV-ICP27-C11 or V27 cells were infected with rHSV-AAV9 at different MOIs (0.2, 0.5, and 1 PFU / cell) using serum-free homemade medium or DMEM supplemented with 2% (v / v) FBS, respectively. The infected cell cultures were incubated in a humidified incubator at 33°C and 5% CO2 for 4-5 days. rHSV-AAV9 was released from the infected cell culture by three freeze-thaw cycles and titrated by plaque assay on V27 cells. On day 2 after infection, the rHSV-AAV9 titer produced by CHO-HV-ICP27-C11 cells was significantly lower than that of V27 cells at MOIs of 0.2, 0.5, and 1 PFU / cell, with the CHO-HV-ICP27-C11 clone producing 5×10 3 , 4×10 4 , and 3.2×10 5 PFU / mL, compared with V27 cells which produced 1×10 6 , 6.75×10 6 and 2×10 6 PFU / mL( Figure 13 ).

[0123] Production of HSV-AAV6.2 vectors in BHK-21-ICP27 cells

[0124] Three recovered BHK21-ICP27 pools were expanded and stored ( Figure 14 ). Aliquots of the pool in 30 ml shake flasks (1×106 viable cells / mL) were tested for HSV-AAV6.2 vector production in the presence of 4% FBS or in serum-free conditions. For cultures supplemented with 4% FBS and 0% FBS, respectively, the percentage of viability of the infected cultures decreased slightly from 87% to 85.5% and 84.4% on day 1 after infection. On day 2 after infection, cell viability decreased to 71.6% and 65.1% for cultures supplemented with 4% FBS and 0% FBS, respectively. On day 3 after infection, cell viability decreased significantly to 45.1% and 44% for cultures supplemented with 4% FBS and 0% FBS, respectively. In addition, for both infected cultures, the viable cell density decreased by an average of 1×106 viable cells / mL per day after infection. 5 cells / ml (data not shown).

[0125] The harvested virus was titrated using a plaque assay on V27 cells. The infected BHK-21-CMV-ICP27 pool supplemented with 4% FBS produced 3.5 × 10 6 PFU / ml, compared to 1.2 × 106 PFU / ml( Figure 15 ).

[0126] Successfully engineered a suspension serum-free adaptive CHO cell pool expressing rHSV-1 invasion and infection of essential HVEM and / or Nectin-1. Compared with wild-type CHO cells, all engineered stable CHO pools all demonstrated a significant susceptibility to rHSV-1-GFP vector invasion and infection, as demonstrated by GFP expression. Compared with the CHO cells infected by the wild type, for five different time points after rHSV-GFP vector infection, the CMV-HVEM pool and CMV-HVEM-CO pool expressing the HVEM gene without codon optimization and through CHO codon optimization were superior to all other pools in GFP expression. These data are consistent with previous announcements, and the announcement reports that engineered CHO expresses the HVEM receptor, allowing HSV-1 to invade and infect (Montgomery et al., Cell Vol. 87, 427-436.).

[0127] Interestingly, pool #1 (referred to as CMV-HVEM) also outperformed all other stable CHO cell pools in terms of producing high rAAV9-GFP vector physical titers at 24 hpi from cell lysis, resulting in the generation of monoclonal cells from this pool, although the difference in titer from the other pools was not significant. Using a high-throughput approach, the top 24 CHO-HVEM expressing clones were selected that showed high growth curves and HVEM expression. These clones were narrowed down to eight clones (C#1, C#13, C#15, C#23, C#24, C#62, and C#64) that showed the highest GFP expression after rHSV-nols-AAV-GFP infection at different time points. These final eight clones were further tested for rAAV6.2-GFP vector production in homemade culture medium at 37°C, starting with an MOI of 1:1 (rHSV-AAV6.2:rHSV-nols-AAV-GFP) for three days. Starting at 24 hpi, both cell viability and viable cell density decreased rapidly. Cell culture media and cell lysates were harvested at 24 and 48 hpi time points and their rAAV6.2-GFP physical titers were tested using Q-PCR. At 24 hpi, the highest rAAV6.2-GFP physical titers were detected in cell lysates from all co-infected clones, followed by a slight decrease in titers at 48 hpi. Lower rAAV6.2-GFP physical titers were detected in culture media from all co-infected clones at different time points (data not shown), indicating that the rAAV6.2-GFP vector is primarily a cell-associated vector from the CHO platform. In addition, harvests at different time points (such as 30 hpi) did not show an improvement in the final rAAV6.2-GFP physical titer compared to harvests obtained at 24 hpi. These findings differ from other systems using AAV-HSV-1-based production, such as HEK293, where the peak of rAAV production is reached 52 hours after co-infection (Kang et al., 2009). Interestingly, the final eight clones tested were variable in HVEM expression. For example, clones (C#1, C#23, and C#24) were high HVEM expressing clones, while clones (C#13, C#15, C#46, C#62, and C#64) were medium HVEM expressing clones, which may have an impact on rAAV production.

[0128] Clones C#1 (designated CHO-HV-C1) and C#62 (designated CHO-HV-C62) produced the highest physical titers of rAAV6.-GFP vector at 24 hpi, so different MOIs were tested on these two clones to improve the final rAAV6.2-GFP titer. MOIs were tested (2:1, 3:1, and 4:1: 6:1, 8:1, and 10:1 from rHSV-AAV6.2 and rHSV-GFP, respectively). MOIs 2:1 and 3:1 did not produce significantly improved titers compared to MOI 1:1. Interestingly, MOI 4:1 produced ~x 10 from clones 10 vg / mL(10 13 vg / L) and ~10 9.37 vg / mL(10 12.37 vg / L); CHO-HV-C1 (high HVEM expression) and CHO-HV-C62 (medium HVEM expression), respectively, showed a positive correlation between HVEM expression and rAAV production. Interestingly, no significant increase in the titer of rAAV6.2-GFP produced using MOI 6:1, 8:1, and 10:1 was detected compared to MOI 4:1 (data not shown). Therefore, CHO-HV-C1 was further selected to test the production of other AAV serotypes, such as AAV8 and AAV9, using different MOIs (such as 1:1, 2:1, 3:1, and 4:1). MOI 1:1, 2:1, and 3:1 produced ~10 8 vg / mL(10 11 vg / L), while MOI 4:1 improved the final titer of rAVV8-GFP and rAAV9-GFP to 10 12.21 and 10 12.40 vg / L. Interestingly, good rAAV8 and rAAV9 titers were detected from cell culture media at 24 hpi using an MOI of 4:1 (data not shown), suggesting that rAAV8-GFP and rAAV9-GFP are not as cell-associated in the CHO platform as rAAV6.2-GFP. Therefore, only low MOIs are required for the production of rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP in the CHO platform, which is in contrast to other studies reporting high MOIs, such as 12:2, as the optimal MOI for rAAV production (Kang et al., 2009).

[0129] Cell viability of CHO-HV-C1 co-infected cells used for rAAV8-GFP and / or rAAV9-GFP vector production decreased dramatically at 24 hpi, similar to the case of rAAV6.2-GFP vector production, indicating that this decrease was not AAV serotype specific. It is believed that the sharp decrease in cell viability after rHSV-1 vector co-infection may affect the final rAAV titer, so the effect of a temperature shift from 37°C to 33°C was tested and showed a significant improvement in cell viability and rAAV6.2-GFP titer after co-infection (data not shown). It has been reported that HSV-1 vectors are 2.5 times more stable at 33°C than at 37°C, and that HSV-1 synchronous infections incubated at 33°C produce twice the amount of vector produced at 37°C (Wechuck et al., 2002). Therefore, co-infection with an MOI of 4:1 (rHSV-1AAV6.2:rHSV-1-nols-AAV-GFP, respectively) was tested at 33°C with shaking at 120 rpm. Interestingly, compared to cultures incubated at 37°C, co-infected cultures at 33°C at 24 hpi showed a slight decrease in cell viability (5-7%), a slight improvement in rAAV6.2-GFP physical titers from lysates, and an approximately two-fold increase in rAAV6.2-GFP titers in the resulting culture medium (data not shown). This finding suggests that shifting the temperature after co-infection to 33°C enhances cell viability and ultimate rAAV6.2-GFP production from the CHO platform.

[0130] Using a self-developed PEG-chloroform purification method followed by Amicon concentration, purified rAAV6.2-GFP and rAAV9-GFP vectors were produced, which showed high efficacy in both in vitro and in vivo studies. This purification method is simple, inexpensive, and rapid for producing rAAV with titers particularly suitable for preclinical studies and is comparable to the more time-consuming iodixanol ultracentrifugation method, and this is consistent with other studies (Wu et al., 2001. Chin. Sci. Bull. 46, 485-488; Negrini et al., Curr Protoc Neurosci. 2020 Sep; 93(1): e103). However, impurities were found in the purified samples during CE-SDS testing. These impurities were confirmed by silver staining of the same samples ( Figure 16 ), suggesting that PEG-chloroform is ideal for preparing rAAV samples for in vitro studies but not for preparing samples for in vivo or clinical studies. Our observations are consistent with recent studies (Kimura et al., Sci Rep. 2019 Sep 19;9(1):13601).

[0131] Analysis of the capsid protein ratios (VP1: VP2: VP3) of different rAAVs (e.g., rAAV6.2-GFP and rAAV9-GFP) produced in CHO-HV-C1 cells using a self-developed CE-SDS method showed that CHO-derived rAAVs had very comparable VP1: VP2: VP3 capsid ratios compared to positive AAV6.2 controls produced by a triple transient transfection system. This suggests that rAAVs produced in CHO-HV-C1 cells are fully packaged, as compared to other platforms that report the need for genetic engineering to enhance VP expression for some AAV serotypes produced in insect cells using a baculovirus system. For example, early experiments using a baculovirus system to adapt AAV-5 for production in insect cells showed low levels of VP1 incorporation into the capsid (Urabe et al., J Virol. 2006; 80: 1874-85; Mietzsch et al., Hum Gene Ther. 2014; 25: 212-22). Interestingly, examination of intact / empty capsids of rAAV produced in the CHO-HV-Cl clone showed a higher percentage of intact capsids than has been previously reported (Small et al., Mol Ther Methods Clin Dev. 2016 May 11;3:16031). Furthermore, testing of CHO cell-derived rAAVs in vitro showed high infectivity titers compared to those produced by triple transient transfection. Furthermore, CHO-derived rAAVs showed very comparable biodistribution in mice compared to those produced by triple transient transfection, particularly for rAAV9-GFP.

[0132] To address the second challenge in scaling up production of rHSV1 vector stocks, CHO-HV-C1 cells were reengineered using random integration and / or CRISPR / Cas9 technology to express rHSV-1 Chinese hamster codon-optimized ICP27 protein. Using homemade culture medium, the final selected clone (called CHO-HV1-ICP27-C11) showed rHSV-1 productive infection, as shown by the replication of the virus propagated on the CHO-HV1-ICP27-C11 clone in V27 cells. However, the production capacity of rHSV-1 vectors from CHO-HV1-ICP27-C11 was lower compared to V27 cells. Compared with the expression of early and mid viral genes, the expression of late HSV-1 viral proteins in infected CHO-HV1-ICP27C11 cells appeared to be little or below normal levels (data not shown). In addition, it seems that CHO cells do not provide the elements necessary for optimal expression of many HSV genes, especially late genes, because CHO cells may express some inhibitory factors that interfere with or block the expression of HSV-1 late viral genes, and our results are consistent with (Shieh et al., J Cell Biol. 1992 Mar;116(5):1273-81). Other factors may be related to the homemade culture medium used, which may have some inhibitory effect on rHSV-1 vector production. Therefore, homemade serum-free adapted BHK-21 cells were engineered to express HSV-1 ICP27 protein and used for the production of rHSV-1 vectors. Interestingly, the stably transfected BHK-21-ICP27 pool produced comparable rHSV-1 titers in Xell medium in the presence or absence of FBS.

[0133] Thus, the present disclosure provides a rAAV-based HSV production platform in engineered CHO cells that provides a scalable, serum-free manufacturing platform that will facilitate the cost-effective manufacture of future rAAV-based biotherapeutics.

Claims

1. A baby hamster kidney cell adapted for growth in serum-free conditions, wherein the cell stably expresses a hamster codon-optimized herpes simplex virus-1 (HSV-1) ICP27 open reading frame comprising a deletion of a non-essential infected cell protein 27 (ICP27) element.

2. The cell of claim 1, wherein the cell is grown in suspension.

3. The cell of claim 1 or 2, wherein the non-essential ICP27 elements are 5' and 3' untranslated regions (UTRs).

4. A cell line comprising the cell according to any one of claims 1 to 3.

5. A method for producing a recombinant adeno-associated virus (rAAV) vector, the method comprising: introducing a recombinant herpes virus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a gene of interest into the cells of any one of claims 1 to 3 or the cell line of claim 4; and culturing the cells or cell line under conditions for producing the rAAV vector.

6. A Chinese hamster ovary (CHO) cell adapted to grow under serum-free conditions, wherein the cell stably expresses one or more polypeptides necessary for the entry and infection of herpes simplex virus-1 (HSV-1).

7. The CHO cell according to claim 5, wherein the CHO cell stably expresses herpes virus entry mediator (HVEM) and / or Nectin-1.

8. The CHO cell according to claim 6, wherein the HVEM and / or nectin-1 sequence has been codon-optimized for expression in CHO cells.

9. A cell line comprising the CHO cell according to claim 5 or 6.

10. A method for producing a recombinant adeno-associated virus (rAAV) vector, the method comprising: introducing a recombinant herpes virus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a gene of interest (GOI) into the cells of any one of claims 5 to 7 or the cell line of claim 8; and culturing the cells or cell line under conditions for producing the rAAV vector.

11. The method of claim 5 or 9, wherein the rHSV vector is introduced at a multiplicity of infection of rHSV-rep / cap:rHSV-GOI of about 4:1, 6:1, 8:1, or 10:

1.

12. The method of any one of claims 5, 9, or 10, wherein the AAV serotype is AAV6, AAV8, or AAV9.

13. The method according to claim 5 or 9, wherein the target gene encodes any therapeutic biological compound.

14. The method of claim 12, wherein the biological compound is an antibody or a chimeric antigen receptor.

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